Figure 3

Functional analyses of Nef–sdAb19 interactions. (A-C) Mutation of G102R/S103E (B) or D60R (A) or the combined triple mutant D60R/G102R/S103E (C) in sdAb19 gradually attenuated binding to Nef as determined by ITC measurements. (D) Mutant sdAb19 proteins abrogate the inhibitory effect of sdAb19 on CD4 internalization. HeLa-CD4 cells were transfected with plasmids for expression of either Nef-GFP or GFP in combination with the plasmid for expression of wild-type or mutated sdAb19 (1:3 Nef:sdAb19 plasmid ratio). Transfected cells were stained with phycoerythrin-conjugated anti-CD4 at 4°C, and the surface expression of CD4 in Nef-GFP- or GFP-expressing cells was measured by flow cytometry (left panel). Results are expressed as the percentage of the mean fluorescence intensity determined in GFP-positive cells relative to that determined in GFP-negative cells. Values are the means from at least 3 independent experiments. Error bars represent 1 standard deviation from the means. Transfected cell lysates were analyzed by Western blot (right panel) using anti-c-Myc and anti-β-actin (top), or anti-GFP (bottom) antibodies.